This application addresses broad Challenge Area (06) Enabling Technologies and Specific Challenge Topic, 06-AG-101: Neuroscience Blueprint: Development of non-invasive imaging approaches or technologies that directly assess neural activity. As the title of the proposal implies (Functional Activity Mapping of Brain Networks), the goal of this proposal is to enable novel technologies for routinely and rapidly mapping behaviorally-driven neural circuits over large regions of the mammalian brain with single cell precision and subcellular detail. Although a number of methodologies have been developed that can map the regional distribution of neural activation within the brain during specific behaviors, knowledge of the cellular participation of circuits involved in behavior-driven activation has been lacking. Because it is known that neural encoding in the cortex involves primarily variations in which neurons are activated by a given event, rather than just how many, it is important to develop methods that enable cellular specification within active circuits. Thus, while non-invasive imaging techniques such as PET and fMRI permit repeated observations of the same subjects and can identify regions exhibiting enhanced activation when learning occurs, what has been missing, until recently, is a method that differentially images functional activity of multiple experiences at cellular resolution. We and our collaborators have developed an approach that allows the determination not only of how many neurons were activated in each of two distinct behavioral episodes in the same animal, but which individual neurons contributed to the encoding of each experience. The method involves a sensitive fluorescence in situ hybridization technique, combined with high resolution confocal microscopy. It also relies on the observations that the expression of the immediate early gene Arc is closely and dynamically coupled to neural activity associated with active information processing, and that the cellular compartmentalization of Arc mRNA varies as a function of time since the activation event (Guzowski et al., 1999). We have called this method "catFISH" (cellular compartment analysis of temporal activity by Fluorescence In Situ Hybridization;Guzowski et al., 1999), because the differential time-course of the post-activity appearance of specific immediate early gene products identifies which neurons were active in each of two separate sessions of behavior. Thus, the activity history of individual cells in a population can be determined for two different time points within the same animal. To achieve our long range goal of whole brain imaging of multiple discrete experiences, with single cell resolution, the present Challenge Grant proposal is focused on overcoming one major impediment for this technology: namely the issue of going from small-scale analysis to more large-scale fully-automated, objective and rapid procedures that will be necessary to implement functional anatomically linked maps of large brain regions. Thus, this proposal focuses on one major Technical Aim, and one Experimental Aim. The first is to develop the software to improve analysis efficiency and objectivity required for projects examining wide areas of the brain, to validate the automated analysis with manual verification to ensure that the methods are robust across brain regions, and to develop novel 3-dimensional montaging methods that will enable automated cytoarchitectonic analysis. These methods should provide a significant step towards the long-range goal of whole brain imaging with cellular resolution, and have the potential to make a significant impact on the range of questions that can be asked about brain circuits by the neuroscience community. Second, the implementation of these automated methods will allow us to examine both hippocampus and large areas of neocortex that would not have been possible within this timeframe with manual methods. Thus, with this tool, we will be able to determine, within an individual animal, whether the degree of context discrimination exhibited by the hippocampus influences the neocortical layers to which the hippocampus projects in young and aged rats. We expect that the outcome of the proposed project will have an important impact on our understanding of the neural basis of episodic memory and how this function changes with normal aging, as well as will benefit the larger neuroscience community's ability to interpret in more detail the activation patterns that are generated by non-invasive imaging methods such as fMRI. PUBLIC HEALTH RELEVANCE: We expect that the outcome of the proposed project will have widespread impact on our understanding of the neural basis of episodic and semantic memory and how this function changes with normal aging. Success of this study will also provide new methods to study memory disorders arising from multiple sources. This technology will be widely disseminated to other investigators in the field.